Improved nucleic acids encoding a chimeric glycosyltransferase

ABSTRACT

The invention relates to nucleic acids which encode glycosyltransferase and are useful in producing cells and organs from one species which may be used for transplantation into a recipient of another species. It also relates to the production of nucleic acids which, when present in cells of a transplanted organ, result in reduced levels of antibody recognition of the transplanted organ.

FIELD OF THE INVENTION

[0001] The present invention relates to nucleic acids which encodeglycosyltransferase and are useful in producing cells and organs fromone species which may be used for transplantation into a recipient ofanother species. Specifically the invention concerns production ofnucleic acids which, when present in cells of a transplanted organ,result in reduced levels of antibody recognition of the transplantedorgan.

BACKGROUND OF THE INVENTION

[0002] The transplantation of organs is now practicable, due to majoradvances in surgical and other techniques. However, availability ofsuitable human organs for transplantation is a significant problem.Demand outstrips supply. This has caused researchers to investigate thepossibility of using non-human organs for transplantation.

[0003] Xenotransplantation is the transplantation of organs from onespecies to a recipient of a different species. Rejection of thetransplant in such cases is a particular problem, especially where thedonor species is more distantly related, such as donor organs from pigsand sheep to human recipients. Vascular organs present a specialdifficulty because of hyperacute rejection (HAR).

[0004] HAR occurs when the complement cascade in the recipient isinitiated by binding of antibodies to donor endothelial cells.

[0005] Previous attempts to prevent HAR have focused on two strategies :modifying the immune system of the host by inhibition of systemiccomplement formation (1,2), and antibody depletion (3,4). Bothstrategies have been shown to prolong xenograft survival temporarily.However, these methodologies are therapeutically unattractive in thatthey are clinically impractical, and would require chronicimmunosuppressive treatments. Therefore, recent efforts to inhibit HARhave focused on genetically modifying the donor xenograft. One suchstrategy has been to achieve high-level expression of species-restrictedhuman complement inhibitory proteins in vascularized pig organs viatransgenic engineering (5-7). This strategy has proven to be useful inthat it has resulted in the prolonged survival of porcine tissuesfollowing antibody and serum challenge (5,6). Although increasedsurvival of the transgenic tissues was observed, long-term graftsurvival was not achieved (6). As observed in these experiments and alsowith systemic complement depletion, organ failure appears to be relatedto an acute antibody-dependent vasculitis (1,5).

[0006] In addition to strategies aimed at blocking complement activationon the vascular endothelial cell surface of the xenograft, recentattention has focused on identification of the predominant xenogeneicepitope recognised by high-titre human natural antibodies. It is nowaccepted that the terminal galactosyl residue, Gal-α(1,3)-Gal, is thedominant xenogeneic epitope (8-15). This epitope is absent in Old Worldprimates and humans because the α(1,3)-galactosyltransferase(gal-transferase or GT) is non-functional in these species. DNA sequencecomparison of the human gene to α(1,3)-galactosyltransferase genes fromthe mouse (16,17), ox (18), and pig (12) revealed that the human genecontained two frameshift mutations, resulting in a nonfunctionalpseudogene (20,21). Consequently, humans and Old World primates havepre-existing high-titre antibodies directed at this Gal-α(1,3)-Galmoiety as the dominant xenogeneic epitope.

[0007] One strategy developed was effective to stably reduce theexpression of the predominant Gal-α(1,3)-Gal epitope. This strategy tookadvantage of an intracellular competition between the gal-transferaseand α(1,2)-fucosyltransferase (H-transferase) for a common acceptorsubstrate. The gal-transferase catalyses the transfer of a terminalgalactose moiety to an N-acetyl lactosamine acceptor substrate,resulting in the formation of the terminal Gal-α(1,3)-Gal epitope.Conversely, H-transferase catalyses the transfer of a fucosyl residue tothe N-acetyl lactosamine acceptor substrate, and generates a fucosylatedN-acetyl lactosamine (H-antigen, i.e., the O blood group antigen), aglycosidic structure that is universally tolerated. Although it wasreported that expression of human H-transferase transfected cellsresulted in high level expression of the non-antigenic H-epitope andsignificantly reduced the expression of the Gal-α(1,3)-Gal xenoepitope,there are still significant levels of Gal-α(1,3)-Gal epitope present onsuch cells.

SUMMARY OF THE INVENTION

[0008] In view of the foregoing, it is an object of the presentinvention to further reduce levels of undesirable epitopes in cells,tissues and organs which may be used in transplantation.

[0009] In work leading up to the invention the inventors surprisinglydiscovered that the activity of H transferase may be further increasedby making a nucleic acid which encodes a H transferase catalytic domainbut is anchored in the cell at a location where it is better able tocompete for substrate with gal transferase. Although work by theinventors focused on a chimeric H transferase, other glycosyltransferaseenzymes may also be produced in accordance with the invention.

[0010] Accordingly, in a first aspect the invention provides a nucleicacid encoding a chimeric enzyme, wherein said chimeric enzyme comprisesa catalytic domain of a first glycosyltransferase and a localisationsignal of a second glycosyltransferase, whereby when said nucleic acidis expressed in a cell said chimeric enzyme is located in an area of thecell where it is able to compete for substrate with a secondglycosyltransferase, resulting in reduced levels of a product from saidsecond glycosyltransferase.

[0011] Preferably the nucleic acid is in an isolated form; that is thenucleic acid is at least partly purified from other nucleic acids orproteins.

[0012] Preferably the nucleic acid comprises the correct sequences forexpression, more preferably for expression in a eukaryotic cell. Thenucleic acid may be present on any suitable eukaryotic expression vectorsuch as pcDNA (Invitrogen). The nucleic acid may also be present orother vehicles whether suitable for eukaryotes or not, such as plasmids,phages and the like.

[0013] Preferably the catalytic domain of the first glycosyltransferaseis derived from H transferase, secretor sialyltransferase, a galactosylsulphating enzyme or a phosphorylating enzyme.

[0014] The nucleic acid sequence encoding the catalytic domain may bederived from, or similar to a glycosyltransferase from an species.Preferably said species is a species such as human or other primatespecies, including Old World monkeys, or other mammals such as ungulates(for example pigs, sheep, goats, cows, horses, deer, camels) or dogs,mice, rats and rabbits. The term “similar to” means that the nucleicacid is at least partly homologous to the glycosyltransferase genesdescribed above. The term also extends to fragments of and mutants,variants and derivatives of the catalytic domain whether naturallyoccurring or man made.

[0015] Preferably the localisation signal is derived from aglycosyltransferase which produces glycosylation patterns which arerecognised as foreign by a transplant recipient. More preferably thelocalisation signal is derived from α(1,3) galactosyltransferase. Theeffect of this is to downregulate the level of Gal-α(1,3)-Gal producedin a cell when the nucleic acid is expressed by the cell.

[0016] The nucleic acid sequence encoding the localisation signal may bederived from any species such as those described above. Preferably it isderived from the same species as the cell which the nucleic acid isintended to transform i.e., if pig cells are to be transformed,preferably the localization signal is derived from pig.

[0017] More preferably the nucleic acid comprises a nucleic acidsequence encoding the catalytic domain of H transferase and a nucleicacid sequence encoding a localisation signal from Gal transferase. Stillmore preferably both nucleic acid sequences are derived from pigs. Evenmore preferably the nucleic acid encodes gtHT described herein.

[0018] The term “nucleic acid” refers to any nucleic acid comprisingnatural or synthetic purines and pyrimidines. The nucleic acid may beDNA or RNA, single or double stranded or covalently closed circular.

[0019] The term “catalytic domain” of the chimeric enzyme refers to theamino acid sequences necessary for the enzyme to function catalytically.This comprises one or more contiguous or non-contiguous amino acidsequences. Other non-catalytically active portions also may be includedin the chimeric enzyme.

[0020] The term “glycosyltransferase” refers to a polypeptide with anability to move carbohydrates from one molecule to another.

[0021] The term “derived from” means that the catalytic domain is basedon, or is similar, to that of a native enzyme. The nucleic acid sequenceencoding the catalytic domain is not necessarily directly derived fromthe native gene. The nucleic acid sequence may be made by polymerasechain reaction (PCR), constructed de novo or cloned.

[0022] The term “localisation signal” refers to the amino acid sequenceof a glycosyltransferase which is responsible for anchoring it inlocation within the cell. Generally localisation signals comprise aminoterminal “tails” of the enzyme. The localisation signals are derivedfrom a second glycosyltransferase, the activity of which it is desiredto minimise. The localisation of a catalytic domain of a first enzyme inthe same area as the second glycosyltransferase means that the substratereaching that area is likely to be acted or by the catalytic domain ofthe first enzyme, enabling the amount of substrate catalysed by thesecond enzyme to be reduced.

[0023] The term “area of the cell” refers to a region, compartment ororanelle of the cell. Preferably the area of the cell is a secretoryorganelle such as the Golgi apparatus.

[0024] In another aspect the invention provides an isolated nucleic acidmolecule encoding a localisation signal of a glycosyltransferase.Preferably the signal encoded comprises an amino terminus of saidmolecule; more preferably it is the amino terminus of gal transferase.The gal transferase may be described from or based on a gal transferasefrom any mammalian species, such as those described above. Particularlypreferred sequences are those derived from pig, mouse or cattle.

[0025] In another aspect the invention relates to a method of producinga nucleic acid encoding a chimeric enzyme said enzyme comprising acatalytic domain of a first glycosyltransferase and a localisationsignal of a second glycosyltransferase whereby when said nucleic acid isexpressed in a cell said chimeric enzyme is located in an area of thecell where it is able to compete for substrate with a secondglycosyltransferase said method comprising operably linking a nucleicacid sequence encoding a catalytic domain from a firstglycosyltransferase to a nucleic acid sequence encoding a localisationsignal of a second glycosyltransferase.

[0026] The term “operably linking” means that the nucleic acid sequencesare ligated such that a functional protein is able to be transcribed andtranslated.

[0027] Those skilled in the art will be aware of various techniques forproducing the nucleic acid. Standard techniques such as those describedin Sambrook et al may be employed.

[0028] Preferably the nucleic acid sequences are the preferred sequencesdescribed above.

[0029] In another aspect the invention provides a method of reducing thelevel of a carbohydrate exhibited on the surface of a cell, said methodcomprising causing a nucleic acid to be expressed in said cell whereinsaid nucleic acid encodes a chimeric enzyme which comprises a catalyticdomain of a first glycosyltransferase and a localisation signal of asecond glycosyltransferase, whereby said chimeric enzyme is located inan area of the cell where it is able to compete for substrate with saidsecond glycosyltransferase, and wherein said second glycosyltransferaseis capable of producing said carbohydrate.

[0030] The term “reducing the level of a carbohydrate” refers tolowering, minimising, or in some cases, ablating the amount ofcarbohydrate displayed on the surface of the cell. Preferably saidcarbohydrate is capable of stimulating recognition of the cell as“non-self” by the immune system of an animal. The reduction of such acarbohydrate therefore renders the cell, or an organ composed of saidcells, more acceptable to the immune system of a recipient animal in atransplant situation or gene therapy situation.

[0031] The term “causing a nucleic acid to be expressed” means that thenucleic acid is introduced into the cell (i.e. bytransformation/transfection or other suitable means) and containsappropriate signals to allow expression in the cells.

[0032] The cell may be any suitable cell, preferably mammalian, such asthat of a New World monkey, ungulate (pig, sheep, goat, cow, horse,deer, camel, etc.) or other species such as dogs.

[0033] In another aspect the invention provides a method of producing acell from one species (the donor) which is immunologically acceptable toanother species (the recipient) by reducing levels of carbohydrate onsaid cell which cause it to be recognised as non-self by the otherspecies, said method comprising causing a nucleic acid to be expressedin said cell wherein and nucleic acid encodes a chimeric which comprisesa catalytic domain of a first glycosyltransferase and a localisationsignal of a second glycosyltransferase, whereby said chimeric enzyme islocated in an area of the cell where it is able to compete for substratewith said second glycosyltransferase, and wherein said secondglycosyltransferase is capable of producing said carbohydrate.

[0034] The term “immunologically acceptable” refers to producing a cell,or an organ made up of numbers of the cell, which does not cause thesame degree of immunological reaction in the recipient species as anative cell from the donor species. Thus the cell may cause a lessenedimmunological reaction, only requiring low levels of immunosuppressivetherapy to maintain such a transplanted organ or no immunosuppressiontherapy.

[0035] The cell may be from any of the species mentioned above.Preferably the cell is from a New World primate or a pig. Morepreferably the cell is from a pig.

[0036] The invention extends to cells produced by the above method andalso to organs comprising the cells.

[0037] The invention further extends to non-human transgenic animalsharbouring the nucleic acid of the invention. Preferably the species isa human, ape or Old World monkey.

[0038] The invention also extends to the proteins produced by thenucleic acid. Preferably the proteins are in an isolated form.

[0039] In another aspect the invention provides an expression unit whichexpresses the nucleic acid of the invention, resulting in a cell whichis immunologically acceptable to an animal having reduced levels of acarbohydrate on its surface, which carbohydrate is recognised asnon-self by said species. In a preferred embodiment, the expression unitis a retroviral packaging cell, cassette, a retroviral construct orretroviral producer cell.

[0040] Preferably the species is a human, ape or Old World monkey.

[0041] The retroviral packaging cells or retroviral producer cells maybe cells of any animal origin where it is desired to reduce the level ofcarbohydrates on its surface to make it more immunologically acceptableto a host. Such cells may be derived from mammals such as canine, rodentor ruminant species and the like.

[0042] The retroviral packaging and/or producer cells may be used inapplications such as gene therapy. General methods involving use of suchcells are described in PCT/US95/07554 and the references discussedtherein.

[0043] The invention also extends to a method of producing a retroviralpackaging cell or a retroviral producer cell having reduced levels of acarbohydrate on its surface wherein the carbohydrate is recognised asnon-self by a species, comprising transforming/transfecting a retroviralpackaging cell or a retroviral producer cell with the nucleic acid ofthe invention under conditions such that the chimeric enzyme isproduced.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 Schematic diagram of normal and chimericglycosyltransferases

[0045] The diagram shows normal glycosyltransferases porcineα(1,3)galactosyltransferase (GT) and human α(1,2)fucosyltransferase(HT), and chimeric transferases ht-GT in which the cytoplasmic domain ofGT has been completely replaced by the cytoplasmic domain of HT, andgt-HT in which the cytoplasmic domain of HT has been entirely replacedby the cytoplasmic domain of GT. The protein domains depicted arecytoplasmic domain CYTO, transmembrane domain TM, stem region STEM,catalytic domain CATALYTIC. The numbers refer to the amino acid sequenceof the corresponding normal transferase.

[0046]FIG. 2 Cell surface staining of COS cells transfected with normaland chimeric transferases

[0047] Cells were transfected with normal GT or HT or with chimerictransferases gt-HT or ht-GT and 48 h later were started withFITC-labelled lectin IB4 or UEAI. Positive-staining cells werevisualised and counted by fluorescence microscopy. Results are from atleast three replicates and values are+/−SEM.

[0048]FIG. 3. RNA analysis of transfected COS cells

[0049] Northern blots were performed on total RNA prepared from COScells transfected: Mock, mock-transfected; GT, transfected withwild-type GT; GT1-6/HT, transfected with chimeric transferase gt-HT;GT1-6/HT+HT1-8/GT, co-transfected with both chimeric transferases gt-HTand ht-GT; HT1-8/GT, transfected with chimeric transferase ht-GT; HT,transfected with normal HT; GT+HT co-transfected with both normaltransferases GT and HT. Blots were probed with a cDNA encoding GT (Toppanel), HT (Middle panel) or g-actin (Bottom panel).

[0050]FIG. 4. Enzyme kinetics of normal and chimericglycosyltransferases

[0051] Lineweaver-Burk plots for α(1,3) galactosyltransferase (□) andα(1,2)fucosyltransferase (▪) to determine the apparent values forN-acetyl lactosamine. Experiments are performed in triplicate, plotsshown are of mean values of enzyme activity of wild-type transferases,GT and HT, and chimeric proteins ht-GT and gt-HT in transfected COS cellextracts using phenyl-B-D Gal and N-acetyl lactosamine as acceptorsubstrates.

[0052]FIG. 5. Staining of cells co-transfected with chimerictransferases

[0053] Cells were co-transfected with cDNAs encoding normal transferasesGT+HT (panels A, B), with chimeric transferases gt-HT+ht-GT (panels C,D), with HT+ht-GT (panels E, F) or with GT+gt-HT (panels G, H) and 48 hlater were stained with FITC-labelled lectin IB4 (panels A, C, E, G) orUEAI (panels B, D, F, H).

[0054]FIG. 6 is a representation of the nucleic acid sequence andcorresponding amino acid sequence of pig secretor.

[0055]FIG. 7 is a representation of the nucleic acid sequence andcorresponding amino acid sequence of pig H.

[0056]FIG. 8 Cell surface staining of pig endothelial cell line (PIEC)transfected with chimeric α(1,2)-fucosyltransferase. Cells weretransfected and clones exhibiting stable integration were stained withUFEAI lectin and visualised by fluorescence microscopy.

[0057]FIG. 9 Screening of chimeric α(1,2)-fucosyltransferase transferasein mice. Mice were injected with chimeric α(1,2)-fucosyltransferase andthe presence of the transferase was analysed by dot blots.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0058] The nucleic acid sequences encoding the catalytic domain of aglycosyltransferase may be any nucleic acid sequence such as thosedescribed in PCT/US95/07554, which is herein incorporated by reference,provided that it encodes a functional catalytic domain with the desiredglycosyltransferase activity.

[0059] Preferred catalytic domains from glycosyltransferase include Htransferase and secretor. Preferably these are based on human or porcinesequences.

[0060] The nucleic acid sequences encoding the localisation signal of asecond transglycosylase may be any nucleic acid sequence encoding asignal sequence such as signal sequences disclosed in P A Gleeson, R DTeasdale & J Bourke, Targeting of proteins to the Golgi apparatus.Glyconjugate J. (1994) 11: 381-394. Preferably the localization sis isspecific for the Golgi apparatus, more preferably for that of the trueGolgi. Still more preferably the localisation signal is based on that ofGal transferase. Even more preferably the localisation signal is basedon porcine, sine or bovine sequences. Even more preferably the nucleicacid encodes a signal sequence with following amino acid sequence (insingle letter code): MNVKGR (porcine), MNVKGK (mouse) or MVVKGK(bovine).

[0061] Vectors for expression of the chimeric enzyme may be any suitablerector, including those disclosed in PCT/US95/07554.

[0062] The nucleic acid of the invention can be used to produce cellsand organs with the desired glycosylation pattern by standardtechniques, such as those disclosed in PCT/US95/07554. For example,embryos may be transfected by standard techniques such as microinjectionof the nucleic acid in a linear form into the embryo (22). The embryosare then used to produce live animals, the organs of which may besubsequently used as donor organs for implantation.

[0063] Cells, tissues and organs suitable for use in the invention willgenerally be mammalian cells. Examples of suitable cells and tissuessuch as endothelial cells, hepatic cells, pancreatic cells and the likeare provided in PCT/US95/07554.

[0064] The invention will now be described with reference to thefollowing non-limiting Examples.

ABBREVIATIONS

[0065] The abbreviations used are bp, base pair(s); FITC, fluoresceinisothiocyanate; GT, galactosyltransferase; H substance, α(1,2)fucosyllactosamine; HT, α(1,2)fucosyltransferase; PCR, polymerase chainreaction;

[0066] EXAMPLE 1

[0067] Cytoplasmic domains of glycosyltransferases play a central rolein the temporal action of enzymes

EXPERIMENTAL PROCEDURES

[0068] Plasmids—The plasmids used were prepared using standardtechniques (7); pGT encodes the cDNA for the porcineα(1,3)galactosyltransferase (23), pHT encodes the cDNA for theα(1,2)fucosyltransferase (human) (25). Chimeric glycosyltransferasecDNAs were generated by polymerase chain reaction as follows: an 1105 bpproduct ht-GT was generated using primers corresponding to the 5′ end ofht-GT (5′-GCGGATCCATGTGGCTCCGGAGCC ATCGTCAGGTGGTTCTGTCAATGC TGCTTG-3′)coding for nucleotides 1-24 of HT (25) followed immediately bynucleotides 68-89 of GT (8) and containing a BamH1 site (underlined) anda primer corresponding to the 3′ end of ht-GT(5′-GCTCTAGAGCGTCAGATGTTATT TCTAACCAAATTATAC-3′) containingcomplementarity to nucleotides 1102-1127 of GT with an Xbal sitedownstream of the translational stop site (underlined); an 1110 bpproduct gt-HT was generated using primers corresponding to the 5′ end ofgt-HT (5′-GCGGATCCATGAATGTCAAAGGAAGACTCTGCCTGGCCT TCCTGC-3′) coding fornucleotides 49-67 of GT followed immediately by nucleotides 25-43 of HTand containing a BamH1 site (underlined) and a primer corresponding tothe 3′ end of gt-HT (5′-GCTCTAGAGCCTCAAGGCTTAG CCAATGTCCAGAG-3′)containing complementarity to nucleotides 1075-1099 of HT with a Xba1site downstream of the translational stop site (underlined). PCRproducts were restricted BamH1/Xba1, gel-purified and ligated into aBamH1/Xba1 digested pcDNA1 expression vector (Invitrogen) and resultedin two plasmids pht-GT (encoding the chimeric glycosyltransferase ht-GT)and pgt-HT (encoding the chimeric glycosyltransferase gt-HT) which werecharacterised by restriction mapping, Southern blotting and DNAsequencing.

[0069] Transfection and Serology—COS cells were maintained in Dubecco'smodified Eagles Medium (DMEM) (Trace Biosciences Pty. Ltd. , CastleHill, NSW, Australia) and were transfected (1-10 μg DNA/5×105 cells)using DEAE-Dextrau (26); 48 h later cells were examined for cell surfaceexpression of H substance or Gal-α(b 1,3)-Gal using FITC-conjugatedlectins: IB4 lectin isolated from Griffonia simplicifolia (Sigma, St.Louis, Mo.) detects Gal-α(1,3)-Gal (27); UEAI lectin isolated from Ulexeuropaeus (Sigma, St. Louis, Mo.) detects H substance (28). H substancewas also detected by indirect immunofluorescence using a monoclonalantibody (mAb) specific for the H substance (ASH-1952) developed at theAustin Research Institute, using FITC-conjugated goat anti-mouse IgG(Zymed Laboratories, San Francisco, Calif.) to detect mAb binding.Fluorescence was detected by microscopy.

[0070] RNA Analyses—Cytoplasmic RNA was prepared from transfected COScells using RNAzol (Biotecx Laboratories, Houston, Tex.), and total RNAwas electrophoresed in a 1% agarose gel containing formaldehyde, the gelblotted onto a nylon membrane and probed with random primed GT or HTcDNA.

[0071] Glycosyltransferase assays—Forty-eight hours after transfection,cells were washed twice with phosphate buffered saline and lysed in 1%Triton X-100/100 mM cacodylate pH 6. 5/25 mM MnCl2, at 4° C. for 30 min;lysates were centrifuged and the supernatant collected and stored at−70° C. Protein concentration was determined by the Bradford methodusing bovine serum allumin as standard (29). Assays for HT activity (30)were performed in 25 μl containing 3 mM [GDP-¹⁴C]fucose (specificactivity 287 mCi/mmol, Amersham International), 5 mM ATP, 50 mM MPS pH6. 5, 20 mM MnCl2, using 2-10 μl of cell extract (approximately 15-20μgof protein) and a range of concentrations (7. 5-75 mM) of the acceptorphenyl-B-D-galactoside (Sigma). Samples were incubated for 2 h at 37° C.and reactions terminated by the addition of ethanol and water. Theamount of ¹⁴C-fucose incorporated was counted after separation fromunincorporated label using Sep-Pak C18 cartridges (Waters-Millipore,Millford, Mass.). GT assays (31) were performed in a volume of 25 μlusing 3 mM UDP[³H]-Gal (specific activity 189 mCi/mmol, AmershamInternational), 5 mM ATP, 100 mM cacodylate pH 6. 5, 20 mM MnCl₂ andvarious concentrations (1 -10 mM) of the acceptor N-acetyl lactosamine(Sigma). Samples were incubated for 2 h at 37° C. and the reactionsterminated by the addition of ethanol and water. ³H-Gal incorporationwas counted after separation from non-incorporated UDP[³H]-Gal usingDowex I anion exchange columns (BDH Ltd., Poole, UK) or Sep-Pak Accellplus QMA anion exchange cartridges (Waters-Millipore, Millford, Mass.).All assays were performed in duplicate and additional reactions wereperformed in the absence of added acceptor molecules, to allow for thecalculation of specific incorporation of radioactivity.

RESULTS

[0072] Expression of chimeric α(1,3)galactosyltransferase andα(1,2)fucosyltransferase cDNAs

[0073] We had previously shown that when cDNAs encodingα(1,3)galactosyltransferase (GT) and α(1,2)fucosyltransferase (HT) weretransfected separately they could both function efficiently leading toexpression of the appropriate carbohydrates: Gal-α(1,3)-Gal for GT and Hsubstance for HT (32). However when the cDNAs for GT and HT weretransfected together, the HT appeared to “dominate” over the GT in thatH substance expression was normal, but Gal-α(1,3)-Gal was reduced. Weexcluded trivial reasons for this effect and considered that thelocalisation of the enzymes may be the reason. Thus, if the HTlocalisation signal placed the enzyme in an earlier temporal compartmentthan GT, it would have “first use” of the N-acetyl lactosaminesubstrate. However, such a “first use” if it occurred, was notsufficient to adequately reduce GT. Two chimeric glycosyltransferaseswere constructed using PCR wherein the cytoplasmic tails of GT and RTwere switched. The two chimeras constructed are shown in FIG. 1: ht-GTwhich consisted of the NH₂ terminal cytoplasmic tail of HT attached tothe transmembrane, stem and catalytic domain of GT; and gt-HT whichconsisted of the NH₂ terminal cytoplasmic tail of GT attached to thetransmembrane, stem and catalytic domains of HT. The chimeric cDNAs weresubcloned into the eukaryotic expression vector pcDNAI and used intransfection experiments.

[0074] The chimeric cDNAs encoding ht-GT and gt-HT were initiallyevaluated for their ability to induce glycosyltransferase expression inCOS cells, as measured by the surface expression of the appropriatesugar using lectins. Forty-eight hours after transfection COS cells weretested by immunofluorescence for their expression of Gal-α(1,3)-Gal or Hsubstance (Table 1 & FIG. 2). The staining with IB4 (lectin specific forGal-α(1,3)-Gal) in cells expressing the chimera ht-GT (30% of cellsstained positive) was indistinguishable from that of the normal GTstaining (30%) (Table 1 & FIG. 2). Similarly the intense cell surfacefluorescence seen with UEAI staining (the lectin specific for Hsubstance) in cells each expressing gt-HT (50%) was similar to that seenin cells expressing wild-type pHT (50%) (Table 1 & FIG. 2). Furthermore,similar levels of mRNA expression of the glycosyltransferases GT and HTand chimeric glycosyltransferases ht-GT and gt-HT were seen in Northernblots of total RNA isolated from transfected cells (FIG. 3). Thus bothchimeric glycosyltransferases are efficiently expressed in COS cells andare functional indeed there was no detectable difference between thechimeric and normal glycosyltransferases.

[0075] Glycosyltransferase activity in cells transfected with chimericcDNAs encoding ht-GT and gt-HT

[0076] To determine whether switching the cytoplasmic tails of GT and HTaltered the kinetics of enzyme function, we compared the enzymaticactivity of the chimeric glycosyltransferases with those of the normalenzymes in COS cells after transfection of the relevant cDNAs. By makingextracts from transfected COS cells and performing GT or HT enzymeassays we found that N-acetyl lactosamine was galactosylated by both GTand the chimeric enzyme ht-GT (FIG. 4. panel A) over a the 1-5 mM rangeof substrate concentrations. Lineweaver-Burk plots showed that both GTand ht-GT have a similar apparent Michealis-Menten constant of Km 2. 6mM for N-acetyl lactosamine (FIG. 4. panel B). Further HT, and thechimeric enzyme gt-HT were both able to fucosylatephenyl-B-D-galactoside over a range of concentrations (7. 5-25 mM) (FIG.4 panel C) with a similar Km of 2. 3 mM (FIG. 4 panel D), in agreementwith the reported Km of 2. 4 mM for HT (25). Therefore the chimericglycosyltransferases ht-GT and gt-HT are able to utilise N-acetyllactosamine (ht-GT) and phenyl-B-D-galactoside (gt-HT) in the same wayas the normal glycosyltransferases, thus switching the cytoplasmicdomains of GT and HT does not alter the function of theseglycosyltransferases and if indeed the cytoplasmic tail is thelocalisation signal then both enzymes function as well with the GTsignal as with the HT signal.

[0077] Switching cytoplasmic domains of GT and HT results in a reversalof the “dominance” of the glycosyltransferases

[0078] The cDNAs encoding the chimeric transferases or normaltransferases were simultaneously co-transfected into COS cells and after48 h the cells were stained with either IB4 or UEA1 lectin to detectGal-α(1,3)-Gal and H substance respectively on the cell surface (Table 1& FIG. 5). COS cells co-transfected with cDNAs for ht-GT+gt-HT (FIG. 5panel C) showed 30% cells staining positive with IB4 (Table 1) but nostaining on cells co-transfected with cDNAs for GT+HT (3%) (FIG. 5 panelA). Furthermore staining for H substance on the surface of ht-GT+gt-Hco-transfectants gave very few cells staining positive (5%) (FIG. 5panel D) compared to the staining seen in cells co-transfected withcDNAs for the normal transferases GT+HT (50%) (FIG. 5 panel B), ie. theexpression of Gal-α(1,3)-Gal now dominates over that of H. Clearly,switching the cytoplasmic tails of GT and HT led to a complete reversalin the glycosylation pattern seen with the normal transferases i.e. thecytoplasmic tail sequences dictate the pattern of carbohydrateexpression observed.

[0079] That exchanging the cytoplasmic tails of GT and HT reverses thedominance of the carbohydrate epitopes points to theglycosyltransferases being relocalized within the Golgi. To address thisquestion, experiments were performed with cDNAs encodingglycosyltransferases with the same cytoplasmic tail: COS cellstransfecterases with cDNAs encoding HT+ht-GT stained strongly with bothUEAI (50%) and IB4 (30%) (Table 1 & FIG. 5 panels E, F) the differencein staining reflecting differences in transfection efficiency of thecDNAs. Similarly cells transfected with cDNAs encoding GT+gt-HT alsostained positive with UEAI (50%) and IB4 (30%) (Table 1 & FIG. 5 panelG, H). Thus, glycosyltransferases with the same cytoplasmic tail leadsto equal cell surface expression of the carbohydrate epitopes, with no“dominance” of one glycosyltransferase over the other observed, andpresumably the glycosyltransferases localised at the same site appear tocompete equally for the substrate.

[0080] In COS cells the levels of transcription of the cDNAs of chimericand normal glycosyltransferases were essentially the same (FIG. 3) andthe immunofluorescence pattern of COS cells expressing the chimericglycosyltransferases: ht-GT and gt-HT showed the typical stainingpattern of the cell space Gal-α(1,3)-Gal and H substance respectively(Table 1 & FIG. 2), the pattern being indistinguishable from that of COScells expressing normal GT and HT. Our studies showed that the Km ofht-GT for N-acetyl lactosamine was identical to the Km of GT for thissubstrate, similarly the Km of gt-HT for phenylBDgalactoside wasapproximately the same as the Km of HT for phenylbDgalactoside (FIG. 3).These findings indicate that the chimeric enzymes are functioning in acytoplasmic tail-independent manner, such that the catalytic domains areentirely functional, and are in agreement with those of Henion et al(23), who showed that an NH₂ terminal truncated marmoset GT (includingtruncation of the cytoplasmic and transmembrane domains) maintainedcatalytic activity and confirmed that GT activity is indeed independentof the cytoplasmic domain sequence.

[0081] If the Golgi localisation signal for GT and HT is containedentirely within the cytoplasmic domains of the enzymes, then switchingthe cytoplasmic tails between the two transferases should allow areversal of the order of glycosylation. Co-transfection of COS cellswith cDNA encoding the chimeric glycosyltransferases ht-GT and gt-HTcaused a reversal of staining observed with the wild typeglycosyltransferases (FIG. 5), demonstrating that the order ofglycosylation has been altered by exchanging the cytoplasmic tails.Furthermore, co-transfection with CDNA encoding glycosyltransferaseswith the same cytoplasmic tails (i. e. HT+ht-GT and GT+gt-HT) gave riseto equal expression of both Gal-α(1,3)-Gal and H substance (FIG. 5). Theresults imply that the cytoplasmic tails of GT and HT are sufficient forthe localisation and retention of these two enzymes within the Golgi.

[0082] To date only twenty or so of at least one hundred predictedglycosyltransferases have been cloned and few of these have been studiedwith respect to their Golgi localisation and retention signals (34).Studies using the elongation transferase N-acetylglucosaminyltransferaseI (33-37), the terminal transferases α(2,6)sialyltransferase (24-26) andβ(1,4)galactosyltransferase (38-40) point to residues contained withinthe cytoplasmic tail, transmembranes and flanking stem regions as beingcritical for Golgi localisation and retention. There are severalexamples of localization signals existing within cytoplasmic taildomains of proteins including the KDEL and KKXX motifs in proteinresident within the endoplasmic reticulum (41,42) the latter motif alsohave been identified in the cis Golgi resident protein ERGIC-53 (43) anda di-leucine containing peptide motif in the mannose-6-phosphatereceptor which directs the receptor from the trans-Golgi network toendosomes (44). These motifs are not present within the cytoplastic tailsequences of HT or GT or in any other reported glycosyltransferase. Todate a localisation signal in Golgi resident glycosyltransferases hasnot been identified and while there is consensus that transmembranedomains are important in Golgi localisation, it is apparent that this isnot essential for the localisation of all glycosyltransferases, as shownby the study of Munro (45) where replacement of the transmembrane domainof α(2,6)sialyltransferase in a hybrid protein with poly-leucine tractresulted in normal Golgi retention. Dahdal and Colley (46) also showedthat sequences in the transmembrane domain were not essential to Golgiretention. This study is the first to identify sequence requirements forthe localisation of α(1,2)fucosyltransferase andα(1,3)galactosyltransferase within the Golgi. It is anticipated thatother glycosyltransferases will have similar localisation mechanisms.

EXAMPLE 2

[0083] Use of secretor in construction of a chimeric enzyme

[0084] A construct is made using PCR and subcloning as described inExample 1, such that amino acids #1 to #6 of the pigα(1,3)-galactosyltransferase (MNVKGR) replace amino acids #1 to 5 of thepig secretor (FIG. 6). Constructs are tested as described in Example 1.

EXAMPLE 3

[0085] Use of pig H transferase in construction of a chimeric enzyme

[0086] A construct is made using PCR and subcloning as described inExample 1, such that amino acids #1 to #6 of the pigα(1,3)-galactosyltransferase (MNVKGR) replace amino acids #1 to 8 of thepig H transferase (FIG. 7). Constructs are tested as described inExample 1.

EXAMPLE 4

[0087] Generation of pig endothelial cells expressing chimericα(1,2)fucosyltransferase

[0088] The pig endothelial cell line PIEC expressing the chimericα1,2fucosyltransferase was produced by lipofectamine transfection ofpgtHT plasmid DNA (20 μg) and pSV2NEO (2 μg) and selecting for stableintegration by growing the transfected PIEC in media containing G418(500 μg/ml; Gibco-BRL, Gaithersburg, Md.). Fourteen independant cloneswere examined for cell surface expression of H substance by stainingwith UEA-1 lectin. >95% of cells of each of these clones were found tobe positive. FIG. 8 shows a typical FACS profile obtained for theseclones.

EXAMPLE 5

[0089] Production of transgenic mice expressing chimericα(1,2)fucosyltransferase

[0090] A NruI/NotI DNA fragment, encoding the full length chimericα1,2fucosyltransferase, was generated utilising the Polymerase ChainReaction and the phHT plasmid using the primers:

[0091] 5′ primer homologous to the 5′ UTR:

[0092] 5′-TTCGCGAATGAATGTCAAAGGAAGACTCTG, in which the underlinedsequence contains a unique NruI site;

[0093] 3′ primer homologous to the 3′ UTR:

[0094] 5′-GGCGGCCGCTCAGATGTTATTTCTAACCAAAT the underlined sequencecontains a NotI site

[0095] The DNA was purified on gels, electroeluted and subcloned into aNruI/NotI cut genomic H-2Kb containing vector resulting in the plasmidclone (pH-2Kb-gtHT) encoding thee chimeric α(1,2)-fucosyltransferasegene directionally cloned into exon 1 of the murine H-2Kb gene,resulting in a transcript that commences at the H-2Kb transcriptionalstart site, continuing through the gtHT cDNA insert. The construct wasengineered such that translation would begin at the initiation condon(ATG) of the hHT cDNA and terminate at the in-phase stop codon (TGA).

[0096] DNA was prepared for microinjection by digesting pH-2Kb-hHT withXhoI And purification of the H-2Kb-hRT DNA from vector byelectrophoretic separation in agarose gels, followed by extraction withchloroform, and precipitation in ethanol to decontaminate the DNA.Injections were performed into the pronuclear membrane of(C57BL/6xSJL)F1 zygotes at concentrations between 2-5 ng/ml, and thezygotes transferred to pseudopregnant (C57BL/6xSJL)F1 females.

[0097] The presence of the transgene in the live offspring was detectedby dot blotting. 5 mg of genomic DNA was transferred to nylon filtersand hybridized with the insert from gtHT, using a final wash at 68° C.in 0.1xSSC/1% SDS. FIG. 9 thaws the results of testing 12 liveoffspring, with two mice having the transgenic construct integrated intothe genome. Expression of transgenic protein is examined by estimatingthe amount of UEAI lectin (specific for H substance) or anti-H mAbrequired to haemagglutinate red blood cells from transgenic mice.Hemagglutination in this assay demonstrates transgene expression.

[0098] It will be apparent to the person skilled in the art that whilethe invention has been described in some detail for the purposes ofclarity and understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

[0099] References cited herein are listed on the following pages, andare incorporated herein by this reference. TABLE 1 EXPRESSION OFGAL-α(1,3)GAL AND H SUBSTANCE BY COS CELLS TRANSFECTED WITH cDNAsENCODING NORMAL AND CHIMERIC GLYCOSYLTRANSFERASES COS cells transfected% IB4 positive % UEAI positive with cDNA encoding: cells cells GT 30 0HT 0 50 ht − GT 30 0 gt − HT 3 50 GT + HT 3 50 ht − GT + gt − HT 33 5GT + gt − HT 30 30 GT + ht − GT 30 0 HT + ht − GT 30 30 HT + gt − HT 050 Mock 0 0

[0100] Transfected COS cells were stained with FITC-labelled IB4 (lectinspecific for Gal-α(1,3)Gal or UEAI (lectin specific for H substance) andpositive staining cells were visualized and counted by fluorescencemicroscopy. Results are from at least three replicates.

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1. A nucleic acid encoding a chimeric enzyme, wherein said chimericenzyme comprises a catalytic domain of a first glycosyltransferase and alocalisation signal of a second glycosyltransferase, whereby when saidnucleic acid is expressed in a cell said chimeric enzyme is located inan area of the cell where it is able to compete for substrate with asecond glycosyltransferase, resulting in reduced levels of a productfrom said second glycosyltransferase.
 2. A nucleic acid according toclaim 1 , wherein said localisation signal localises said catalyticdomain thereby to enable the catalytic domain to compete with saidsecond glycosyltransferase for a substrate.
 3. A nucleic acid accordingto claim 1 or claim 2 , wherein the localisation signal is derived froma glycosyltransferase which produces glycosylation patterns which arerecognised as foreign by a transplant recipient.
 4. A nucleic acidaccording to any one of claims 1 to 3 , wherein the localisation signalcomprises the amino terminus of the second glycosyltransferase.
 5. Anucleic acid according to any one of claims 1 to 4 , wherein thelocalisation signal is derived from α(1,3)-galactosyltransferase.
 6. Anucleic acid according to any one of claims 1 to 5 , wherein the firstglycosyltransferase is selected from the group consisting ofH-transferase, secretor sialyltransferase, a galactosyl sulphatingenzyme or a phosphorylating enzyme.
 7. A nucleic acid according to anyone of claims 1 to 6 , wherein the catalytic domain and the localisationsignal each originates from a mammal selected from the group consistingof human, primates, ungulates, dogs, mice, rats and rabbits.
 8. Anucleic acid according to any one of claims 1 to 7 , wherein thelocalisation signal is derived from the same species as the cell whichthe nucleic acid is intended to transform.
 9. A nucleic acid accordingto any one of claims 1 to 8 , comprising a sequence encoding thecatalytic domain of H transferase and a nucleic acid sequence encoding alocalisation signal from Gal transferase.
 10. A nucleic acid accordingto claim 9 , wherein the catalytic domain and the localisation signalare derived from pigs.
 11. A nucleic acid according to any one of claims1 to 10 , which encodes gtHt as defined herein.
 12. A vehicle comprisinga nucleic acid according to any one of claims 1 to 11 .
 13. vehicleaccording to claim 12 , selected from the group consisting of anexpression vector, plasmid and phage.
 14. A vehicle according to claim12 or claim 13 , which enables said nucleic acid to be expressed inprokaryotes or in eukaryotes.
 15. An isolated nucleic acid moleculeencoding a localisation signal of a glycosyltransferase.
 16. An isolatednucleic acid molecule according to claim 15 , wherein the signal encodedcomprises an amino terminus of gal-transferase.
 17. A method ofproducing a nucleic acid according to any one of claims 1 to 11 ,comprising the step of operably linking a nucleic acid sequence encodinga catalytic domain from a first glycosyltransferase to a nucleic acidsequence encoding a localisation signal of a second glycosyltransferase.18. A method of reducing the level of a carbohydrate exhibited on thesurface of a cell, said method comprising causing a nucleic acid to beexpressed in said cell wherein said nucleic acid encodes a chimericenzyme which comprises a catalytic domain of a first glycosyltransferaseand a localisation signal of a second glycosyltransferase, whereby saidchimeric enzyme is located in an area of the cell where it is able tocompete for substrate with said second glycosyltransferase, and whereinsaid second glycosyltransferase is capable of producing saidcarbohydrate.
 19. A method of producing a cell from a donor specieswhich is immunologically acceptable to a recipient species by reducinglevels of carbohydrate on said cell which cause it to be recognised asnon-self by the recipient, said method comprising causing a nucleic acidto be expressed in said cell wherein said nucleic acid encodes achimeric enzyme which comprises a catalytic domain of a firstglycosyltransferase and a localisation signal of a secondglycosyltransferase, whereby said chimeric enzyme is located in an areaof the cell where it is able to compete for substrate with said secondglycosyltransferase, and wherein said second glycosyltransferase iscapable of producing said carbohydrate.
 20. A cell produced by a methodaccording to claim 19 .
 21. An organ comprising a cell according toclaim 20 .
 22. A non-human transgenic animal, organ or cell comprisingthe nucleic acid according to any one of claims 1 to 11 .
 23. Anexpression unit which expresses a nucleic acid according to any one ofclaims 1 to 11 , resulting in a cell which is immunologically acceptableto an animal having reduced levels of a carbohydrate on its surface,which carbohydrate is recognised as non-self by said species.
 24. Anexpression unit according to claim 23 , selected from the groupconsisting of a retroviral-packaging cassette, retroviral construct orretroviral producer cell.
 25. A method of producing an expression unitaccording to claim 23 or claim 24 , said unit having reduced levels of acarbohydrate on its surface wherein the carbohydrate is recognized asnon-self by a species, comprising transforming/transfecting a retroviralpackaging cell or a retroviral producer cell with the nucleic acid ofthe invention under conditions such that the chimeric enzyme isproduced.